Compounds from biopolymers and effector substances which are linked via optically active amino acid derivatives, processes for the preparation thereof and the use thereof

ABSTRACT

The invention relates to compounds from biopolymers and effector substances which are linked with the aid of derivatives of optically active amino acids in which the amino group has been converted into a maleimido group and the carboxyl group into an active ester group.

The invention relates to compounds from biopolymers and effectorsubstances which are linked with the aid of derivatives of opticallyactive amino acids in which the amino group has been converted into amaleimido group and the carboxyl group into an active ester group.

Conjugates which can be prepared from biopolymers such as peptides orproteins and marker or effector substances or specific binding partnersby covalent chemical linkage are needed in the diagnostic andtherapeutic field. Preferred methods which are employed in thesecouplings are those in which the specific properties of the componentsto be linked to each other are not changed at all or else are changed ina defined way by the coupling. A summary of known methods for thepreparation of protein conjugates from the field of enzyme immunoassaysis given in a review article by Ishikawa et al. (J. Immunoassay 24(1983) 209-327). Methods for the preparation of immunotoxins are, forexample, listed in a review article by Ghose et al. (Methods inEnzymology, Vol. 93 (1983) pp. 280-333). Processes for the preparationof conjugates using heterobifunctional reagents make possible thespecific controlled linkage of the coupling partners. In the case ofprotein conjugates, methods in which linkages of SH groups with aminogroups are established play an important role because of the low risk ofside reactions if the reaction is carried out in a proper way.Cross-linking reagents having maleimido, haloalkyl or haloacyl andactive ester structures are preferably used in this case.

The cross-linking reagents which have been described so far were notsuitable, since they have no chiral carbon atom in the bridge, for theformation of conjugates with a defined chiral structure in the bridge.Moreover, it has not been possible in the case of cross-linking reagentshaving a short-chain bridging component to suit the polarity of thecross-linking agent to the particular conditions required.

The object on which the present invention is based was now to provideconjugates from biopolymers and effector substances, which are importantfor diagnosis and therapy, in a sterically defined form, it beingnecessary to suit the cross-linking reagent, with respect to length,mobility and polarity, to the optimum coupling conditions for thecomponents to be coupled. Furthermore, the bridges formed duringcoupling are to be of sufficient stability and, if appropriate,cleavable in a predetermined fashion.

The present invention now relates to compounds of the formula I ##STR1##in which *C is an asymmetric carbon atom, R is the side chain of anatural amino acid, of methionine sulfone or of cysteic acid,

X is the radical of the thiol coupling component and

Y represents the radical of the amino coupling component.

Compounds in which the thiol coupling component X-SH and the aminocoupling component Y-NH₂ are each proteins are preferred here. Compoundsin which

X--SH is an enzyme, an antibody or a fragment of an antibody and

Y--NH₂ is an enzyme, an antibody or a fragment of an antibody areparticularly preferred.

Compounds in which the enzyme is peroxidase, β-galactosidase orphosphatase, and the antibodies or the fragments of antibodies arederived from rabbits, sheep, goats or mice, are very particularlypreferred, antibodies or fragments of antibodies against CEA, AFP, HCG,TSH and HBsAg being particularly preferred.

Compounds in which R is the side chain of the amino acid alanine, ofmethionine sulfone or of cysteic acid, and very particularly preferrablyof the amino acid alanine are furthermore preferred.

The invention also relates to a process for the preparation of acompound according to formula I, in which process an amide is formed ina first reaction step by reacting an amino component Y--NH₂ with thefunctional active ester group of a compound of the formula II ##STR2##in which *C is an asymmetric carbon atom, R has the meaning stated forformula I and R¹ is hydrogen or a radical of the formula III ##STR3## inwhich R³ is hydrogen or C₁ to C₄ alkyl, and a thioether is formed in asecond reaction step by Michael addition, by reacting a thiol componentX--SH with the maleimido group of the compound of the formula II.

The invention further relates to compounds of the formula II in which Rand R¹ have the meanings stated therefor.

Compounds preferred in this case are those in which R corresponds to theside chain of the amino acid alanine, of methionine sulfone or ofcysteic acid, and very particularly of the amino acid alanine.

The invention also relates to the use of compounds according to formulaI for analysis, diagnosis or therapy. The use in an enzyme immunoassayis preferred in this connection.

The invention finally relates to the use of a compound according toformula II for the preparation of a compound of the formula I. For thepreparation of the linker reagents according to formula II, maleimidogroups are formed, for example by processes known to those skilled inthe art, by reacting primary amino groups with maleic anhydride andcyclizing the maleoyl derivatives formed with the elimination of waterto give maleimido derivatives (maleoyl derivatives) (Keller and Rudinger(1975); Helvetica Chimica Acta 58, 531-541 and Rich et al. (1975); J.Med. Chem. 18, 1004-1010).

The N-hydroxysuccinimide or sulfo-N-hydroxysuccinimide ester groups areformed, for example, from the carboxyl groups and N-hydroxysuccinimideor the sulfone derivative thereof with the elimination of water and withthe aid of reagents such as carbodiimides (Anderson et al. (1964); J.Am. Chem. Soc. 86, 1839 et seq.).

If the side chains R contain groups which undergo side reactions underthe conditions for formation of the maleimido group or the active estergroups, appropriate substitutions or modifications must be carried outthere to prevent these side reactions. In reagents derived from theamino acid methionine, the oxidation-sensitive thioether functionalitiesare, for example, converted into sulfone groups which, in addition,bring about better solubility in water of the resulting compound. In thecase of cysteine, a SH-protecting group which is readily soluble inwater, such as the acetamidomethyl (Acm) protective group, is, forexample, employed, or, even more advantageously, an oxidation of the SHgroup to give a sulfonic acid group with the formation of cysteic acidwhich, as the sulfonic acid salt, can be employed for the synthesis ofthe corresponding heterobifunctional reagent. In the case of lysine orornithine, it is preferred to use polar, acid-stable amino-protectivegroups such as the methylsulfonyloxycarbonyl (Msc) group as theprotein-modification reagents. If appropriate, the protective group forthe amino acid side chain is selected such that, after linking the firsttwo coupling partners, the protective groups on the side chain can beeliminated without destroying the properties of the coupling partnersrequired for the application. The use of the Msc protective group asamino-protective group or of the thiopyridyl radical for the protectionof thiol are examples. Both protective groups can be removed in manycases of protein-protein conjugates without harming the proteins. In thecase of protection of thiol, the liberated thiol functionality can beused for introducing a third coupling component.

The compounds according to formula II can be employed for thepreparation of conjugates by methods known to those skilled in the art.A survey of methods for the preparation of protein conjugates in thefield of enzyme immunoassays is, for example, given in the reviewarticle by Ishikawa et al. (J. Immunoassay 4 (1983) 209-327). Methodsfor the preparation of immunotoxins are, for example, listed in thereview by Ghose et al. (Methods of Enzymology, vol. 93 (1983) pp.280-333).

Reagents which contain a chiral carbon atom in the bridge between thetwo functional groups have not been described for use in immunoassays. Aderivative containing the structure --CH(CH₃)--CO-- in the bridgepresents itself as the simplest member of this series. Such a reagentcontaining maleimido and succinimide ester or sulfosuccinimide estergroups is, as expected, readily soluble in water because of the smallhydrophobic portion in the molecule. It can be prepared from the aminoacid alanine as described above.

Reagents with the structure II can advantageously be used for linkingimmunoglobulins and marker enzymes for immunoassays.

In this case, thiol groups can for example be introduced into theimmunoglobulin (Ishikawa et al., 1983), maleimido groups can beintroduced into the marker enzyme by reacting the amino groups of theenzyme and, after the removal of excess reagents,the modifiedimmunoglobulin can be reacted with the modified enzyme for coupling.

In another way of carrying out the reaction, maleimido groups can beintroduced by reacting the amino groups of the immunoglobulin withreagents of the structure II and can be reacted with thiol groupsalready present in the marker enzyme (e.g. β-galactosidase from E.coli). or with thiol groups which have been introduced by reaction withreagents such as 2-iminothiolane, for coupling.

The conjugations can be carried out in a particularly specific fashionwith respect to the binding site and also to stoichiometry if the hingethiols in Fab' of immunoglobulins are reacted with maleimido groups inthe enzyme which can be obtained by reacting the enzyme with reagents ofthe structure II. The coupling of the enzyme is then carried out on theend of the Fab' opposite the paratope. In an analogous case, thiolgroups can be generated by reducing agents in the hinge region ofimmunoglobulins and can be reacted with the maleimido groups of theenzyme. It was surprising that the amino groups of one protein could bereadily linked with the SH groups of the amino acid cysteine in theother protein in view of the short length and low flexibility in thebridging component of the cross-linking reagents.

The examples which follow are intended to illustrate the preparation ofcompounds of the structure II and the use thereof for the preparation ofcompounds of the structure I.

EXAMPLE 1 Preparation of maleoyl-L-alanine

L-alanine (26.7 g; 0.30 mol) is added to a stirred solution of maleicanhydride (29.4 g; 0.30 mol) in glacial acetic acid (300 ml). Afterstirring for 3 hours, the reaction product is filtered off, dried andrecrystallized from acetone/hexane. Further crystals can be obtained onconcentrating the mother liquor. The total yield is 33 g (60%). Themelting point of the product is 142-144 degree C.

EXAMPLE 2 Preparation of maleoyl-L-alanine

Maleoyl -L-alanine (4 g; 21 mmol) is suspended in dry toluene (150 ml),triethylamine (4 ml) is added and the mixture is then boiled, withstirring, for 6 h under reflux with a water trap. The hot solution isthen decanted off the orange colored residue and, after cooling, isconcentrated to dryness in a rotary evaporator. 50 ml of water which hasbeen adjusted to pH 3 with hydrochloric acid are added to the viscousresidue which is extracted 5 times at pH 3 with 50 ml of ethyl acetatein each case, readjusting the pH each time. The combined ethyl acetatephases are concentrated to dryness in a rotary evaporator and the solidproduct obtained is dried overnight under high vacuum. Yield: 1.25 g.

¹ H-NMR (200 MHz, CDCl₃ ; TMS): δ 1.65 (d, 3H, I=7.4 Hz, CH₃), 4.82 (q,1H, I=7.4 Hz, CH), 6.74 (s, 2H, maleimido), 8.30 (bs, COOH).

EXAMPLE 3 Preparation of maleoyl-L-alanine-N-hydroxysuccinimide ester(MAS)

Maleoyl-L-alanine (200 mg; 1.2 mmol) and N-hydroxysuccinimide (150 mg;1.3 mmol) are dissolved in tetrahydrofuran. A solution ofdicyclohexylcarbodiimide (255 mg; 1.2 mmol) in tetrahydrofuran (1 ml) isadded dropwise and while stirring to the solution which has been cooledto 0° C., and stirring is continued at 4° C. overnight. The resultingsuspension is filtered off and the filtrate is concentrated to drynessin a rotary evaporator. The residue is resuspended in 2 ml oftetrahydrofuran and the suspension is filtered through a P4 glass frit.The filtrate is concentrated to dryness in a rotary evaporator and thesolid product obtained is dried under high vacuum. Yield: 330 mg.

¹ H-NMR (200 MHz, CDCl₃ ; TMS): δ 1.77 (d, 3H, I=7.4 Hz, CH₃), 2.84 (s,4H, succinimidyl) 5.17 (q, 1H, I=7.4 Hz, CH), 6.80 (s, 2H, maleimido).

EXAMPLE 4 Preparation of a maleimido-antibody a) Reagents

MAS which has been prepared according to Example 3 is used; boric acid(E. Merck, order No. 165); dioxane E. Merck, order No. 3110); lithiumhydroxide (E. Merck, order No. 11652).

b) Preparation of solutions

Lithium borate buffer, pH 8.5

Boric acid (1.24 g) is stirred in a mixture of water (80 ml) and dioxane(20 ml). The pH is adjusted to 8.5 by the addition of solid lithiumhydroxide while dissolving the boric acid.

MAS solution

MAS (0.0124 g) is dissolved in dioxane (1 ml).

Phosphate buffer pH 6.0

Sodium dihydrogen phosphate monohydrate (41.4 g) and Titriplex (5.58 g)are dissolved in water (3 1). The pH is adjusted to 6.0 with sodiumhydroxide solution.

c) Preparation of the maleimido-antibody

The antibody to be conjugated (concentration 4 g/l in 1/15 Mphosphate-buffered physiological saline, 25 ml) is mixed with lithiumborate buffer (25 ml). 0.45 ml of MAS solution (corresponding to a30-fold molar excess of MAS over IgG) is added to the resulting solutionhaving a pH of 7.5, while stirring. After incubation at room temperaturefor 1 hour, the excess reagents are removed by gel filtration on aSephadex G-25 column equilibrated with phosphate buffer, pH 6.0.

EXAMPLE 5 Preparation of a thiol-peroxidase a) Reagents

Peroxidase (degree of purity I, Boehringer Mannheim, order No. 815462),2-iminothiolane hydrochloride (Sigma, order No. 6256), methanol (E.Merck, order No. 6009), disodium tetraborate decahydrate (E. Merck,order No. 6308)

b) Solutions

Sodium borate solution

Disodium tetraborate (0.952 g) is dissolved in water (100 ml).

Iminothiolane solution

2-Iminothiolane (0.688 g) is dissolved in methanol (5 ml).

c) Preparation of the thiol-peroxidase

Peroxidase (160 mg) is dissolved in sodium borate buffer and mixed withiminothiolane solution. After incubation of the reaction mixture at roomtemperature for 2 hours, the excess reagents are removed by gelchromatography on a Sephadex G-25 column equilibrated with phosphatebuffer, pH 6.0 (Example 4, section b)).

EXAMPLE 6 Preparation of a peroxidase/antibody conjugate a) Reagents

Maleimido-antibody and thiol-peroxidase according to Example 4) andExample 5), TRIS (E. Merck, order No. 9382).

b) Solutions

TRIS buffer, pH 7.4

TRIS (18.17 g) is dissolved in water (3 1) and the pH is adjusted to 7.4with HCl.

c) Preparation of the peroxidase/antibody conjugate

Maleimido-antibody solution and thiol-peroxidase solution are mixedcorresponding to a molar ratio of antibody to peroxidase of 1:5. Afterincubation at room temperature for 2 hours, the reaction is stopped byaddition of 1/10 volumes of NEM solution. Excess reagents are removedafter incubation for 30 minutes by gel chromatography on a Sephadex G-25column which has been equilibrated with TRIS buffer.

d) Properties of the conjugate

No remaining free antibody is detected on HPLC analysis by gelchromatography on a DuPont GF-250 column. A molar ratio of peroxidase toantibody of 2.5 ±0.5 on comparison of various preparations withpolyclonal antibodies is found in the conjugate peak by opticalanalysis. In the case of monoclonal antibodies, the molar ratio can varyin the range from 1.5 l to 3.5 l. The conjugate can be used directly forthe enzyme immunoassay in the dilution used specifically for theparticular assay. Fractionation of the conjugate by gel chromatographyimproves the specific reaction of the conjugate in a few cases.

EXAMPLE 7 Preparation of a maleimido-peroxidase a) Reagents andsolutions

N-Ethylmorpholine, 98% pure (Riedel de Haen, order No. 62050),N,N-dimethylformamide (Riedel de Haen, order No. 15440), all otherreagents and solutions as in Examples 4-6.

b) Preparation of the maleimido-peroxidase

Peroxidase (1 g) is dissolved in water (6.5 ml ). Dioxane (3.5 ml) ismixed into this solution. The pH is adjusted to 8.5 withethylmorpholine. This solution is mixed with a solution of MAS (24 mg)in dimethylformamide (0.5 ml). After incubation at room temperature for1 hour the buffer is changed to phosphate buffer, pH 6.0, by gelfiltration on Sephadex G-25.

EXAMPLE 8 Preparation of Fab'--SH

Fab' with free SH groups in the hinge region is prepared by knownmethods (Ishikawa et al. (1983) J. Immunoassay 4, 209-327). For this,IgG is cleaved to give F(ab')₂ using pepsin. The disulfide bridges ofthe hinge in the F(ab')₂ are reduced to thiols by thiol/disulfideexchange using mercaptoethylamine. The reaction mixture is freed ofexcess thiol and the buffer is changed to phosphate buffer, pH 6.0 bygel filtration.

EXAMPLE 9 Preparation of a Fab'/peroxidase conjugate

Fab'--SH according to Example 8 (100 mg in 20 ml of phosphate buffer, pH6.0) is mixed with maleimido-peroxidase according to Example 7 (500 mgin 10 ml of phosphate buffer, pH 6.0) and the mixture is incubated at 37degrees Celsius for 1 hour. The conjugate formed is separated from theremaining Fab'--SH and excess peroxidase by gel chromatography onUltrogel ACA 44 (from Pharmacia). TRIS buffer, pH 7.4, is the eluent. Inthe case of rabbit antibodies, the main portion of the conjugate elutescorresponding to a 1+1 adduct of Fab' and peroxidase in the gelchromatography. The product can be used directly for the enzymeimmunoassay in the dilution used specifically for the assay.

We claim:
 1. A compound of the formula II ##STR4## in which *C is anasymmetric carbon atom, R is the side chain of a natural amino acid, ofmethionine sulfone or of cysteic acid, and R¹ is hydrogen or a radicalof the formula III ##STR5## in which R³ is hydrogen or C₁ to C₄ alkyl.2. The compound as claimed in claim 1, in which R corresponds to theside chain of the amino acid alanine, methionine sulfone or cysteicacid.
 3. The compound as claimed in claim 2, in which R corresponds tothe side chain of the amino acid alanine.